Almost all the bones of mammals are formed through a mechanism called “endochondral bone formation” in which chondrocytes calcify via the growth and differentiation thereof, and are finally replaced with bone. It is known that a variety of hormones and growth factors participate in this series of process, including insulin-like growth factor (IGF1, IGF2), fibroblast growth factor (FGF), transforming growth factor (TGF), growth hormone and the like. Hiraki et al. isolated ChM-I gene as a factor, in addition to the above hormones and growth factors, that facilitates the growth and differentiation of chondrocytes (Biochem. Biophys. Res. Commun., 175, 971-977, 1991, European Patent Publication No. 473080). Human ChM-I is synthesized as a type II membrane protein comprising 334 amino acid residues and, after glycosylation, undergoes processing with a result that the C-terminal portion comprising 120 amino acid residues are extracellularly secreted (Hiraki et al., Eur. J. Biochem. 260, 869-878, 1999). ChM-I not only promotes the growth of cultured chondrocytes but potently promotes proteoglycan synthesis and the colony formation of chondrocytes in agarose (Inoue et al., Biochem. Biophys. Res. Commun., 241, 395-400, 1997). ChM-I also promotes the growth of osteoblasts (Mori et al., FEBS Letters, 406, 310-314, 1997).
On the other hand, it has long been pointed out that cartilage remains not only avascular but resistant to infiltration of blood vessels. Hiraki et al. attempted to purify a growth inhibiting factor for vascular endothelial cells from the extracts of cartilaginous tissue, and have succeeded in the complete purification thereof. As a result, it was found to be ChM-I (Hiraki et al., FEBS Letters, 415, 321-324, 1997; Hiraki et al., J. Biol. Chem., 272, 32419-32426, 1997). Generally, the cartilaginous tissue is characterized by being avascular, but in the replacement to the bone tissue, it is believed, infiltration of blood vessels into the cartilaginous tissue is required. In the scheduled region of vascular invasion, the hypertrophy of cartilaginous tissue and the calcification of cartilage matrix occur prior to vascular invasion to be ready for forming the primary point of ossification. In the region where the hypertrophic cartilage and the subsequent ossified cartilage appear, the expression of ChM-I dramatically decreases. Thus, although the expression of the ChM-I gene is cartilage-specific, it is limited to the avascular cartilage that is resistant to vascular invasion. As described above, it is believed that ChM-I not only promotes the growth, differentiation, and maturing of cartilage but inhibits the infiltration of blood vessels by inhibiting the growth of vascular endothelial cells. Thus, the expression in the avascular cartilage and the disappearance of expression in the ossified layer prior to vascular invasion are in good agreement with the bifunctional effect of ChM-I.
In the cartilaginous tissue, bFGF that is a potent angiogenic factor is accumulated in pericellular space in large quantities, and it has been elucidated that ChM-I is present in interterritorial space in such a way as to surround bFGF (Hiraki et al., J. Biol. Chem., 272, 32419-32426, 1997). Thus, in the avascular cartilage, ChM-I is present in a form that masks the angiogenic factor, and it is thought that the angiogenenic effect of ChM-I may account for the absence of blood vessels in the cartilage (Tanpakusitsu kagaku koso, Vol. 40, No. 5, 1995). It has also been confirmed that ChM-I suppresses the growth of tumor cells by inhibiting the infiltration of blood vessels into human tumor cells in vivo (Hayami et al., FEBS Letters, 458, 436-440, 1999). The expression analysis of ChM-I in various mouse tissues revealed that ChM-I is expressed in the eye and the thymus in addition to the cartilage, but the function of ChM-I in these tissues has yet to be elucidated (Shukunami et al., Int. J. Dev. Biol. 43, 39-49, 1999).
The growth and the expression of differentiation function of chondrocytes plays an important role in the healing process from fracture or various cartilage diseases. Thus ChM-I, a factor that promotes the growth and differentiation of chondrocytes, is a promising candidate for an agent that promotes the growth of chondrocytes (Kokai (Japanese Unexamined Patent Publication) No. 7-138295). In the growth or metastasis of tumor cells, infiltration of blood vessels into tissues is required to obtain energy necessary therefor. Therefore ChM-I that has an effect of inhibiting angiogenesis is also a likely candidate for an anti-cancer agent (Kokai (Japanese Unexamined Patent Publication) No. 7-138295). As described above, ChM-I not only controls the growth and differentiation of chondrocytes but inhibits angiogenesis, and hence its application into drugs is being awaited.
In recent years, biotechnology has made rapid progress, and in association with the development of the human genome project as well, a great number of new genes are being cloned. It is said that the number of human genes amounts to about 100,000, and among the genes groups of molecules having a homology in the amino acid sequences sometimes form families. As the groups of molecules having a homology in the amino acid sequences, various gene families are known such as the TNF family, the TNF receptor family, the chemokine family, G-protein coupled receptor family and many other gene families. For example, as the molecules belonging to the TNF family, there are known about 20 molecules including tumor necrosis factor α (TNFα, Pennica et al., Nature 312, 724, 1984), Fas ligand (FasL, Suda et al., Cell 75, 1167, 1993), TNF-related apoptosis-inducing ligand (TRAIL, Steven et al., Immunity 3, 673, 1995), B lymphocyte stimulator (BLYS, Moore et al., Science 285, 260-263, 1999), and the like.
The molecules belonging to the TNF family are type II membrane proteins and have a homology in the amino acid sequence in the extracellular region. Although homology in the amino acid sequence is noted, these molecules have been demonstrated to have their inherent functions, and their application as pharmaceutical agents have been attempted in a variety of diseases. It has also been shown that the molecules of the TNF family have their unique receptors, and the application thereof as pharmaceutical agents have also been attempted. In fact, some have been approved as pharmaceutical products (for example, soluble TNF receptor, by Immunex). Research and development is also in progress on antibodies against these molecules as pharmaceutical drugs, and in fact, some have been approved as pharmaceutical products (for example, anti-TNF-α antibody, by Centocore). As examples of molecules having a homology in the amino acid sequence that were applied into the development of pharmaceutical products, the TNF family and the TNF receptor family were illustrated as above. Some of the underlying reasons that enabled the application of these molecules into pharmaceutical products are the facts that the functions of each of these molecules were analyzed and the similarity and the difference between them were elucidated.
Molecules of the TNF family have the structure of type II membrane proteins and since many of them are expressed mostly in the blood system and the lymphatic system, they have a lot in common in terms of experimental techniques and samples. It is therefore expected that when a new gene belonging to the TNF family was discovered, the speed at which its function was analyzed must have been faster than the molecules discovered earlier. Thus, the discovery of a novel gene having a homology in the amino acid sequence and the analysis of its function would not only facilitates the functional analysis of novel genes to be discovered in the future but the result of analysis permits its comparison with the existing molecules, and therefore it is expected that more detailed findings on the functions of the existing molecules could be obtained.
Generally, when a novel gene encoding a protein having a homology in the amino acid sequence with existing molecules is cloned, the techniques and materials to be used for functional analysis may be referred to the examples of the existing molecules. However, even a molecule having a homology in the amino acid sequence is thought to have its own unique function as in the above-mentioned TNF family, and thus when its application into pharmaceutical products is envisaged, it is necessary to demonstrate the expression and purification of the recombinant protein, the generation of antibody, the expression of mRNA and protein at various tissues and the like, and thereby to clarify the difference in the structure and function from the existing molecules.